The goal of the proposed research is to identify those structural features of iron-binding proteins which underlie their biological activity, and relate these to molecular mechanisms in the binding, release and functional activity of iron. Of central interest is the role of the transferrins, a class of two-sited iron binding proteins ubiquitously distributed in cells and physiologic fluids of vertebrates, in the transport and delivery of iron. Kinetic, spectroscopic and physiologic methods will be applied to the study of serum transferrin, in efforts to understand the detailed features of iron uptake from the protein by cells. A possible role of the transferrin receptor, which remains complexed to transferrin throughout the transferrin-cell cycle, in facilitating release of iron from protein to cell will be explored. Kinetic studies of iron release in vitro will be correlated with ENDOR (electron nuclear double resonance) and ESEEM (electron spin echo envelope modulation) spectroscopic analyses to explore the roles of conformational changes and the specific anion-binding site of transferrin in the binding and release of iron from each site of the protein. Studies of single-sited half-transferrins, proteolytically cleaved from the native protein, will facilitate analysis by eliminating the confounding effects of simultaneous release from each site as well as iron exchange between sites. Related experiments will compare the iron- donating functions of the two sites in efforts to account for their population differences in the circulation. Ferritin, an iron-storage protein in species ranging from microbes to man, may hold up to 4500 iron atoms in its hollow core. Early events in the accumulation of iron by ferritin will be spectroscopically studied to ascertain whether histidine serves as a way station in the accumulation of iron by the ferritin core. The binuclear iron center of uteroferrin, a prototype of the class of purple acid phosphatases with binuclear metal centers, still eludes understanding. Of particular interest are the interactions of the spin- coupled binuclear iron center with ortho-phosphate, the product of the protein's enzymic activity. After selective replacement of the two distinguishable iron atoms with diamagnetic Zn(II) or with 57Fe, EPR, ENDOR, ESEEM and NMR spectroscopic studies will examine how each iron atom and its ligands respond to phosphate and related perturbing anions.